Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching. Author Nicole Taylor, Ming-Tzo Wei, Howard Stone, Clifford Brangwynne Publication Year 2019 Type Journal Article Abstract Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research. Keywords Humans, HEK293 Cells, Caenorhabditis elegans Proteins, RNA Helicases, Fluorescence Recovery After Photobleaching Journal Biophys J Volume 117 Issue 7 Pages 1285-1300 Date Published 2019 Oct 01 ISSN Number 1542-0086 DOI 10.1016/j.bpj.2019.08.030 Alternate Journal Biophys J PMCID PMC6818185 PMID 31540706 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML